GB2496122A - Biological sample preservation on paper - Google Patents

Abstract

The invention discloses a cellulose or glass fibre paper for preservation of biological samples, which comprises 4 40 30 wt % of a hydrophilic braned carbohydrate polymer. The polymer may be Ficoll (RTM) or dextran, either of which may be bound to the surface such as by reaction with epichlorohydrin. Also disclosed is a method for preservation of biological samples such as dried blood spots or proteins by applying and drying them on the paper.

Description

INTELLECTUAL

. .... PROPERTY OFFICE Applicalion No. GBI 1 18731.7 RTM Dale:17 April2012 The following terms are registered trademarks and should be read as such wherever they occur in this document: Ficoll Intellectual Properly Office is an operaling name of Ihe Patent Office www.ipo.gov.uk

SAMPLE PRESERVATION METHOD AND SAMPLE PRESERVATION SUBSTRATE

Technical field of the invention

The present invention relates to methods for samp'e preservation, and more particularly to sample preservation on paper substrates. The invention also relates to paper substrates for sample preservation and to methods of manufacturing such substrates.

Background of the invention

Increasing use is being made of paper substrates in the analysis andior storage of biological materials. One such area of use concems the growing need for rapid analysis of large quantities of blood samples in pharmacokinetic research, for example in drug discovery programmes. It is obviously desirable for such uses that the paper combines satisfactory mechanical properties with an ability to hold the biological material of interest in such a way that it can be subjected to analysis and/or thrther processing following storage. Examples of such papers are those known as FTA and FTA Elute (Whatman, part of GE Healthcare) described for example in US Patents Nos. 5,756,126 and 5,939,259. These papers have been impregnated with chemicals to provide cell lysis, preservation of nucleic acids and to facilitate further processing of nucleic acids.

However, these papers are specifically designed for nucleic acid analysis. There is also a strong interest in the use of paper substrates for processing of samples where proteins are analysed. Proteins differ from nucleic acids i.a. in that they arc prone to denaturation and consequent loss of biological activity upon drying and storage. Proteins to be analysed, e.g. biomarkers or drugs in blood, are also often present in very low amounts and prone to masking by additives. In particular, analytical techniques such as mass spectroscopy and immunoassays can be affected by chemical additives. Plain untreated cellulose papers such as the 903 or 31 ETF papers (Whatman, part of GE Healthcare) are used for preservation of blood samples followed by protein analysis, but do not always give the desirable analytical recoveries and biological activites of e.g. sensitive proteins. Addition of different fillers such as polyvinyl alcohol, Ficoll and sugars has been mentioned as a way to stabilize proteins in e.g. US2OIO/0209957 and W02003/020924. These compositions are however aLso limited in the recoveries and activities of sensitive proteins.

Accordingly there is a need for sample preservation methods and sample preservation substrates with improved performance.

Summary of the invention

One aspect of the invention is to provide sample application substrate capable of receiving a liquid biological sample and providing a high recovery of biologically active proteins after drying and storage. This is achieved with a paper as defined in claim 1.

One advantage is that a high recovery of sensitive proteins can be obtained. Further advantages arc that homogenous circular sample spots can be obtained and that liquid samples arc well absorbed by the substrate.

Another aspect of the invention is to provide a sample collection device capable of receiving a sample and providing a high recovery of biologically active proteins afler drying and storage.

This is achieved with a sample collection device as defined in claim 12.

A third aspect of the invention is to provide a method of applying a sample to a sample applicatioa substrate which provides a high recovery of biologically active proteins after drying and storage. This is achieved by a method as defined in claim 14.

A fourth aspect of the invention is to provide a method of preparing a paper substrate capable of receiving a sample and after drying and storage providing a high recovery of biologically active proteins. This is achieved with a method as defined in claims 21 and 22.

Further suitable embodiments of the invention are described in the dependent claims.

Definitions The term "paper" as used herein means a fibrous web or sheet material. Paper comprises fibres, e.g. cellulose or glass fibres, and optionally other components, such as e.g. particulate fillers, wet strength or dry strength additives, retention agents etc. The term "carbohydrate polymer" as used herein means a polymer with a main chain comprising carbohydrate moieties. A carbohydrate polymer can be a potysaccharide (i.e. a polymer with a main chain consisting of linked carbohydrate moieties), a modified polysaccharide (i.e. a polysaccharide with substituents or grafted side chains) or a synthetic carbohydrate polymer, where carbohydrate moieties are linked to each other via synthetic linking units. A hydrephilic carbohydrate polymer is in this context either water soluble or water swellable, wherein in the latter case the polymer may be prevented from dissolving in water by crosslinks or by tethers to one or more surfitces.

The term "analyte" as used herein means a substance undergoing or intended to undergo detection, quantification, analysis, characterisation and/or evaluation.

The term "contaminant" as used herein means a substance having the potential to interfere with the detection, quantification, analysis, eharacterisation or evaluation of one or more analytes. An analyte can also be a contaminant if it has the potential to interfere with the detection, quantification, analysis, characterisation or evaluation of another analyte.

The term "sample" as used herein means a portion of a fluid, liquid, semisolid or solid material.

The term "ligand" as used herein means a chemical species capable of binding or attracting another species. If a ligand is attached to a solid surface, dissolved substances may bind to or be retained by thc surface, depending on thc selectivity of thc ligand for each substance.

Detailed descriotion of embodiments In one aspect the present invention discloses a paper useful as a sample application substrate for preservation ofbiological samples. The paper has a surface weight of 40 -800 g/m2, it coiipiises cellulose fibres and/or glass fibres, and it also comprises 4-30 wt % of a hydrophilic or water soluble branched carbohydrate polymer. The surface weight is determined by weighing an air-dry sheet of predetermined area of the paper (e.g. 10.0 x 10.0 cm) and dividing the weight (g) with the sheet area (m2). The branched carbohydrate polymer can e.g. have a degree of branching of 0.05-1, such as 0.10-1,0.20-1 or 030-1. The degree of branching (DB) is defined as DB = nD/(nD + L), where D is the number of branch-point monomer units in a polymer molecule, n is the average number of branches extending from each branch-point and L is the number of linear (non-branching) monomer units in the polymer molecule. The degree of branching can be determined according to methods known in the art, e.g. by NMR spectroscopy, degradation analysis, and by gel filtration using a light scaftering or viscosity detector. (K Granath: J Coll Sci 13, 308 1958; J Smit et al: Macromolecules 25, 3585 1992). Examples of branched carbohydrate polymers include dextrans, Ficoll and branched hemicclluloscs such as xylans. An advantage of having a carbohydrate polymer in the paper is that it provides a protective effect on proteins, possibly by preventing dcnaturation. Branched carbohydrate polymers have particularly good protective effect and they are also easier to apply due to their lower viscosities. An advantage of having at least 4% of thc polymer in the paper is that good protection is obtained over this lcvcl. An advantage of having no more than 30% polymcr in the paper is that thc spots formed by application of liquid biological samples, e.g. blood, on the paper are circular and homogeneous below this level. Ifthe polymer content is too high, the spot shape will be irregular, causing difficulties in the subsequent sampling from the paper. The absorption of the samples into the paper is also better if the polymer content does not exceed 30%. At too high polymer contents, a blood spot will not be completely absorbed by the paper, so that a film of dried blood is formed on the paper surface. This means that part of the dried blood is not in contact with the preserving polymer and there is also a risk of losing chips of dried blood so that a quantitative sampling is not possible. The combination of protective effect and spot shape/homogeneity/absorption is particularly good if the amount of the branched carbohydrate polymer in the paper is 10 -25 wt %, such as 12-25 wt%.

In some embodiments, the hydrophilic or water solublc branched carbohydrate polymer has an average molecular weight of 15 -800 lcDa, such as 20-500 kDa. It can in particular have an averagc molecular weight of 70-400 kDa, such as about 70 kDa or about 400 kDa. Thc protective effect is better if the molecular weight is not too low and with very high molecular weights, viscosity can become an issue both during impregnation of the paper and during extraction of the proteins from the paper.

In some embodiments, the branched carbohydrate polymer comprises a dextran. Dextrans are branched a (1-.6)-linked glucans and the most common variety, produced from sucrose solutions by the Lcuconostoc mesenteroides NRRIL B-512(F) bacteria, have side chains attached to the 3 positions of the backbone glucose units with approx. 5% of the glucose units forming branching points. This gives a degree of branching of 0.05 as defined above. 10 wt % aqueous dextran solutions have a Newtonian rheology, with viscosities about 2 mPas (Mw 10 kDa), 4 mPas (40 kDa) and 10 mPas (70 kDa). B-S 12(F) dextrans arc commercially available from e.g. Pharmacosmos A'S (Denmark) or TdB Consultancy AB (Sweden). It is also possible to use dextran derivatives such as e.g dextran sulfate, carboxymethyl (CM) dextran or diethylaminoethyl (DEAE) dextran. Such derivatives are available from e.g. TdB Consultancy AB.

In certain embodiments, the branched carbohydrate polymer comprises a copolymer of a mono-or disaccharide with a bifunctional epoxide reagent. Such polymers will be highly branched due to the multitude of reactive hydroxyl groups on each mono/disaccharide.

Depending on the reaction conditions used, the dcgrec of branching can be from about 0.2 up to almost 1. A particular example of these polymers is sucrose-epiehlorohydrin polymers, prepared e.g. according to the methods of US3300474. A commercially available sucrose-epichlorohydrin polymer is FicollM, which is also called polysucrose (CAS No. 25702-74-3).

It is available from GE Healthcare (Sweden) in average molecular weights of 70 and 400 kDa.

wt % aqueous solutions of Ficoll have a Newtonian rheology, with a viscosity of about 3 mPas (70 kDa) and about S mPas (400 kfla). The low viscosities are presumably due to the highly branched structure of the polymer molecules. Charged varieties of Fico II such as CM Ficoll and DEAE Ficoll can be obtained e.g. from TdB Consultancy AB (Sweden) In some embodiments, a 10 wt % water solution of the branched carbohydrate polymer has a viscosity of 1 -10 mPas, such as 1-5 mPas, at 20°C. A low viscosity is advantageous from several points of view. It facilitates impregnation of the base paper and ensures that the distribution of the polymer in the paper is homogeneous. It also facilitates the extraction and further handling of the extracted solutions. Low viscosities may also be beneficial in that thc sample is absorbed more easily by the paper and in that the paper does not get sticky.

In certain embodiments, the content of water extractables in said paper isO -25 wt%, such as 0.1 -5 wt % or 3-20 wt %. The amount of water extraetables is determined by immersing a piece of the paper in water (water-paper volume ratio at least 20:1) and agitating gently at room temperature for 2 h. The paper is then collected on e.g. a glass filter, dried and weighed.

The water extractables are calculated as the % weight loss. Water extractable amounts less than about 1 % may be determined by e.g total organic carbon analysis of the water extract after filtration through a glass fiber filter free from extractables. Having a low amount of water extractables can be an advantage when analytical techniques sensitive to the presence of carbohydrate polymers or other extractables are used for analysis of the protein(s). Very low amounts of extractables can be achieved when the carbohydrate polymer is covalently coupled to the paper fibres and/or crosslinked to itself In some embodiments, the paper comprises 5-300 micromole/g, such as 5-50, 5-100 or 50-300 micromole/g negatively or positively charged groups. Negatively charged groups can be e.g. carboxylate groups, sulibnate groups or sulfate groups, while positively charged groups can be e.g. amine or quaternary ammonium groups. The presence of these groups appears to improve the protective effect of the branched carbohydrate polymer. The negatively or positivcly charged groups can be covalently bound to the carbohydrate polymer or they can be covalently bound to the cellulose or glass fibres of the base paper. If the negatively charged groups are bound to the ares or to a carbohydrate polymer which is crosslinked or covalently coupled to the fibres, the charged groups are not water extractable. This can be an advantage, particularly if mass spectroscopy is used as a method of analysis. Amounts of negatively or positively charged groups can be determined e.g. by well-known titration methods.

Additionally, sulfonate, sulfate, amine and quaternary ammonium groups can be determined by elementary analysis, provided that non-charged sulfur or nitrogen containing species are not present.

In certain embodiments the paper comprises at least one dried biological sample, such as a dried blood sample. Blood and other biological materials, e.g. serum, plasma, urine, ccrcbrospinal fluid, bone marrow, biopsies etc. can be applied to the paper and dried for storage and subsequent analysis.

In one aspect the present invention discloses a sample collection device, coiiqrising the paper as described above. The sample collection can be a paper card, with one or more sample application areas printed or otherwise indicated on the card. There may be indicator dyes in these areas to show if a non-coloured sample has been applied or not. The device may also include a card holder, to e.g. facilitate automatized handling in racks etc. and it may include various fbrms of sampling features to facilitate the collection of the sample.

In one aspect the present invention discloses a method for preservation of at least one biological sample, comprising the steps of: a) providing a biological sample, b) applying said biological sample on the paper described above, and c) drying said paper with said biological sample.

The biological sample may be a biological fluid, e.g. blood, serum, plasma, urine, cerebrospinal fluid, bone marrow etc. but it may also be a solid or semi-solid such as tissue biopsies etc. An advantage of applying a fluid sample is that it is absorbed by the paper structure and comes into direct contact with the protective branched carbohydrate polymer.

The drying can be passive, when the paper with the sample is simply left to dry, or it can be active, when it is subjected e.g. to moderately elevated temperatures, infrared radiation and/or a stream of air or other gas. Thc drying can advantageously be performed at tcmperatures of -35 °C, such as 15-25 °C. After drying, the residual moisture content of the paper, or a sample application area with a sample, can be less than about 20 wt. %, such as less than about lOwt.%.

In certain embodiments the method comprises a step d) of storing the dried paper with the biological sample for at least one week, such as at least one month or at least one year. The storage temperature in step d) can be at least 0°C, such as 0-40°C or 20-40°C. It is an advantage of the method of the invention that the structure and biological activity of sensitive proteins can be maintained over long times without the need of freezing or even without refrigeration. The paper with the sample(s) can advantageously be stored at a relative humidity between 0 and 70%, such as between 0 and 50% or between 0 and 20%. The relative humidity can e.g. be controlled by storing the paper with the samples together with a desiccant in a closed container, such as a closed plastic bag.

In some embodiments the method comprises a step e) of extracting at least one protein from said paper after storage and analyzing said protein. The extraction can be made e.g. by punching out small parts of the paper with dried sample and immersing these in an aqueous liquid, e.g. a buffer for a period of e.g. 1 h -48 h. The immersion may be performed under agitation and the temperature may e.g. be 0°C to 30°C. If the immersion time is more than 1 - 2 h, it is an advantage if the immersion temperature isO-8°C to minimize any degradation of sensitive proteins in solution.

In certain embodiments the protein is a storage-sensitive protein, which can be defined as a protein giving less than about 60%, or less than about 40%, recovery of the protein in a biologically active state after storage in plain paper with a desiccant for I week at 37 °C. This value can preferably be determined with the protein being present in a dried blood spot (DBS) on the paper. One example of a storage-sensitive protein is C-reactive protein (CRP), but also proteins such as apolipoprotein A-i, IgE have been reported as being sensitive to storage in DBS (T tvlcDadc et at: Demography 44, 899 2007).

In some embodiments in step e) of the method, the recovery of said protein in a biologically active state is at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.

In some embodiments the protcin is analyzed in step c) by an immunoassay, by mass spectrometry or an enzyme activity assay. Immunoassays, e.g. ELISA, are commonly used to determine the concentration of particular proteins. As they rely on the antibody-binding properties of the protein it is essential that the protein is biologically active (i.e. not denatured), in particular when assays with antibodies against conformation epitopcs are used.

Enzymes are generally sensitive to denaturation and a denatured enzyme will show no activity or a strongly reduced activity in an assay. Mass spectrometry (MS) is another common analysis technique for proteins, which requires that the sample applied to the mass spectrometer does not contain leachables from the paper, e.g. detergents. The branched polysaccharidc polymers used can easily be removed from the sample by conventional work-up methods for MS analysis, e.g. reversed phase chromatography, and hence they do not produce interference.

In one aspect the present invention discloses a method of manufacturing a paper for preservation of biological samples. Thc mcthod comprises thc following steps: a) provide cellulose fibres or glass fibres either in an aqueous suspension or in the form of a base paper, b) contact the fibres, either in suspension or as a base paper, with a solution comprising 2-60 wt % of a water soluble branched carbohydrate polymer, c) if the fibres are in aqueous suspension, form a paper sheet from the fibres, and d) dry the paper.

The contacting of a fibre suspension with the polymer solution can take place simply by mixing the suspension and the solution or by dissolving the polymer in the suspension. The contacting of a base paper with the polymer solution can take place as an impregnation step as described below. If the polymer is to be eovalently coupled, the solution can have a concentration of e.g. 20-60 wt% polymer, while in the case of impregnation of a base paper with a branched carbohydrate polymer under conditions not giving covalent coupling, the concentration can be e.g. 2-15 wt% or 4-10 wt%. Formation of a paper sheet can be done according to methods well known in the art, either by handsheet formation or by formation on a paper machine, e.g. a mould or Fourdrinier machine. The drying can be passive, when the paper with the sample is simply left to dry, or it can be active, when it is subjected e.g. to elevated temperatures, infrared radiation and/or a stream of air or other gas. The drying can advantageously be performed at temperatures of 20 -100 °C, such as 20 -60 °C. After drying, the residual moisture content of the paper, can be less than about 20 wt. %, such as less than about 10 wt. %. In a production setting, the drying can suitably be made in an on-line dryer applied e.g. after an immersion coater or the formation unit of a paper machine.

In one aspect the present invention discloses a method of manufacturing a paper for preservation of biological samples. The method comprises the following steps: a) provide a base paper having a surface weight of 40-800 g/m2, which comprises cellulose fibres or glass fibres, b) impregnate the base paper with a solution which comprises 2 -60 wt % of a water soluble branched carbohydrate polymer with an average molecular weight of 15 -800 kDa c) dry the impregnated paper.

The impregnation can be made by immersing a sheet of base paper in the solution or it can be made in a roll-to-roll process using various types of impregnation equipment known in the art, such as e.g. immersion coaters. If the polymer is to be covalently coupled, the solution can have a concentration of e.g. 20-60 wt% polymer, while in the case of impregnation with a branched carbohydrate polymer under conditions not giving covalent coupling, the concentration can suitably be e.g. 2-15 wt% or 4-10 wt%. The drying can be passive, when the paper with the sample is simply left to dry, or it can be active, when it is subjected e.g. to elevated temperatures, infrared radiation and/or a stream of air or other gas. The drying can advantageously be performed at temperatures of 20 -100 °C, such as 20-60°C. After drying, the residual moisture content of the paper, can be less than about 20 wt. %, such as less than about 10 wt. %. In a production setting, the drying can suitably be made in an on-line dryer applied e.g. after an immersion eoater.

In certain embodiments the branched carbohydrate polymer comprises dextran or a copolymer of a mono-or disaccharide with a bifunctional epoxide reagent as described above. The branched carbohydrate polymer can specifically be a sucrose-epichiorohydrin polymer, such as Ficoll. The branched carbohydrate polymer can have an average molecu'ar weight of 20- 500 kDa or 70-400 kDa, such as 20 kDa, 70 kDa or 400 kDa.

In some embodiments the solution of the branched carbohydrate polymer has a viscosity of I -20 mPas, such as 1 -10 mPas at 20°C. As stated above, low viscosity solutions facilitate impregnation and also bring about further functional advantages.

In certain embodiments the base paper comprises 5-300 micromole/g negatively charged groups, such as carboxylate groups. The negatively charged groups may be introduced in the paper e.g. by oxidation processes or by covalent coupling of charged species, such as chloroacetic acid, bromoacetic acid, sodium vinylsulphonate etc. according to known techniques for preparing po lysaceharide-based cat ion exchangers.

In certain embodiments the fibres or the base paper are chemically activated and the method comprises a step of reacting the fibres or the base paper with the branched carbohydrate polymer. An advantage of using activated fibres/base paper is that a covalent coupling of the carbohydrate polymer can be achieved. This has the advantage that interference of extracted polymer with analysis methods can be avoided. Having a covalently coupled polymer can also be beneficial for the preservation of proteins. The activation can be accomplished by several different methods, e.g. by reaction with an epihalohydrin (e.g. epichlorohydrin) or a diepoxide, in which case the activated fibres or the activated paper will comprise reactive epoxy groups. Alternatively, the fibres/the paper can be activated by tosylation, tresylation or mesylation, creating the corresponding reactive leaving groups, or they can be activated with divinylsulphone or by allylation followed by either bromination of the allyl groups or direct coupling on the allyls, etc. It is advantageous if the activated groups are reactive towards hydroxyl groups in the branched carbohydrate polymer, but they can also be reactive to specific thnetional groups introduced in the polymer, e.g. amines or aminooxy groups (reactive e.g. towards aldehydes) or carboxyl groups (reactive towards carbodiimides) etc. Glass fibres can be activated by reaction with silanes, e.g. epoxy silanes. The reaction between the carbohydrate polymer and the activated fibres/paper can take place directly in the suspension, optionally after heating andlor adjusting pH to accelerate the reaction. It can also take place during or after drying of the paper, e.g at elevated temperature.

In certain embodiments the method also comprises a step of washing the fibres or the base paper after the reaction step. This removes unreacted polymer from the system and is a further way to ensure that the polymer does not leach out and interfere with any analysis methods.

The washing can suitably take place after the reaction described above.

Examples

Materials 31 ETF base paper (Whatman, part of GE Heal thcare) 903 base paper (Whatman, part of GE Healthcare) Dextran T3.5 (3.5 kDa), Dextran 20 (20 kDa), Dextran 70 (70 kDa), Dextran 110 (110 kDa) (GE Healthcare) Ficoll PM2O (20 kDa), Ficoll PM7O (70 kDa), Ficoll PM400 (400 kDa) (GE Healthcare) DEAF Ficoll PM7O (70 kDa), CM Ficoll PM7O (70 kDa) (ThE Consultancy AB) Example 1 -Soaking in Ficoll and dextran solutions Strips were cut from the chosen paper, the air-dry weight recorded and the paper placed in 90mm plastic Petri dishes, two strips in each. The desired aqueous solution (25mL) of Ficoll or dextran was added and the dish placed on a shaker and agitated at 100rpm for lh 45mm.

The prototypes were then removed from the Petri dishes and placed horizontally in a Whatman 903 Dry Rak and allowed to dry at room temperature overnight. The weight was recorded and thc increase in mass calculatcd. Finally the prototypes were tagged with a plastic label and stored in 90mm Petri dishes for ifirther DBS analysis.

Table 1 Weight increase/or the prepared prototypes, samples in duplicates çprnpound Concentration in solution (wt %) Weight increase of paper (wt %) FicollPM400 10 25 Ficol! PM400 7 15 Ficoll PM400 5 12 Lofl PM400 2 4 Fic.oll PM7O 1 0 26 Fico!1 PM7O 7 16 Fico]! PM70 5 11 Ficoll PM70 2 3 Ficoll PM7O 1 1 Dcxtran 110 10 27 Dextran 11 0 7 17 Dextran 110 5 12 IJextran 110 2 4 Dcxtran 110 1 1 Dextran 20 10 26 Dextran20 7 18 Dextran20 5 12 Dcxtran 20 2 4 Dcxtran 20 1 2 Dextran T3.5 10 26 DextranT3.5 7 18 DextranT3.5 5 12 Dcxtran T3.S 2 4 Dextran T3.5 1 2 The mass increase for all prototypes is very similar, for example when a 10 wt% solution has been used the weight increase is 25-27% regardless which polymer has been used.

Example 2 -Covalent coupling of Ficoll and dextran Epichlorohydrin(ECH) couphng of Ficoll PM7O In a typical experiment a paper strip was cut from 31 Etf paper, which had been placed at 60°C and dried at least overnight prior to the experiment. Plastic nets were cut to cover the inside of lSOmL glass reactor and the paper strip was sandwiched between them. To the reactor water(103.2mL), 50%NaOH(10.6mL) and ECH(16.lmL) were added and the solution stirred for 120mm at 30°C. The reaction mixture was decanted and l3OmL of water added and stirring continued for 20mm. This was repeated twice.

Ficoll PM7O solution(lOOmL) with desired concentration was added to the reactor and the solution stirred for 15 minutes prior to the addition of 50%NaOH(2.91 6mL). The reaction was stirred at 30°C overnight. After 20h the reaction mixture was decanted and I 30m L of water added and stirring continued for 20mm. The paper prototype was removed from the reactor and placed in a glass beaker(ôOOmL) and water(300mL) was added covering the prototype completely. The beaker was slowly agitated on a shaker at 80rpm for 20mm after which the water was decanted and fresh water added. This was repeated three times. The paper was left to dry in the ifime hood overnight on plastic nets to ensure the prototype was flat. The prototypes were stored in Zip-Lock bags until analysed further.

ECH coupling of FicoR PM7O on repulped fibre In a typical experiment about 1 5g of 31 ETF paper was torn to small pieces and thrown into a blender together with water(-500m1) and mixed harshly for about 3mm into a pulp. The pulp slush was poured into a sintered glass funnel (P2) and most of the water removed. The weight was recorded 80g) and the slightly moist pulp was transferred to a 500mL round bottom flask (RBE). To the RBF water( 142mL) and 50%NaOH(25niL) were added and the mixture stirred for 15 mm using an overhead stirrer. Whilst stirring ECFI(3OmL) was added and the water bath set to 30°C. The mixture was then stirred for additionally 2h.

The reaction was terminated by transferring the pulp to a sintered glass funnel and washing it with 5x Water. The pulp was stirred afler each wash and the suction started when the water startcd to drop through thc funnel.. The drained pulp was then transferred to a ncw 500mL RBF which was charged with Fieoll PM7O solution(25mL). The mixture was stirred for -20mm before addition of 50%NaOH(8.7SmL). Stirring was continued overnight at 30°C.

After about 20h the reactions were terminated and the fibre prototype washed on a glass filter using 5x Water. This turned out to be a quite tedious procedure especially for the high concentration fibres. Ta the last washing step the still fairly wet fibre was transferred to a Zip-Lock bag and used for handsheet paper production. Unmodified 3lEtf fibre was also prepared and used for handsheet production.

Soaking and ECH coupling of Ficoll PM2O and soaking of Dextran 70 Experiments were performed as described above with some alterations for the coupled Ficoll PM2O prototypes.

Smaller glass reactors (lOOmL) were used and the reaction volumes decreased; for the activation water(23.83mL), 50%NaOH(2.4SniL) and ECH(3.72mL) was used. The coupling was performed using Ficoll PM2O solution(3OmL) and 50%NaOH(744.8tL).

The coupled DEAE-Ficoll PM7O prototypes were sent for elemental analysis (%N), Table 2.

According to the analysis certificate provided by TdB Consultancy the Nitrogen content of DEAE-Ficoll PM7O is 3.6%, so a 0.086% N contcnt in the paper should correspond to a DEAE-Ficoll PM7O content of 2.4 wt%.

Table 2 Elemental analysis qf the DEA E-Fic:oli prototypes (%N,) Coupling Prototype Paper Ligand.

Solution(wt /o) (g/g) U2612068E 31Eff DEAE-FicoII 20 <0.01 U2612068F 3lEtf DEAE-FicoII 50 0,086 Example 3 -Recovery of C-reactive protein from dried blood spots B'ood samples (4p.l!spot) were spotted on various types of soaked and cova.lent.ly coupled prototypes and reference papers. After drying spots were punched out and extracted according to conditions used in previous studies (section 2). The amount of CRP and total protein was determined by ELISA and A280 respectively. The recovery is calculated from the amount in reference blood samples (n3).

Spotting and storage of DBS samples 4 R1 blood aliquots were spotted on the different prototypes in triplicates DBS dried in RT for 4 hand stored (-20 C) with drying agent in sealed bags until use. 4 p1 blood aliquots were stored in 0.5 ml tubes for reference purpose (stored -20°C).

DBS extraction Blood spots were punched in triplicate with punch pliers. The size was chosen to punch complete spot (5 mm diameter). The discs were put in 96-well plates and extracted with 100 pA PBS-T during shaking (400 rpm), lh RT, over night -I-4 C, lh RT. The plate was sealed during incubation and centrifuged at 3700 rpm in plate centrifuge prior to sample analysis.

CRP ELISA / A280 measurement The Human CRP ELISA Kit was used according to manufactures instruction.

Reference blood aliquots (n=3) were diluted with 100 p1 PBS-T. References and extracted samples were then further diluted 1:10 in CRP wash buffer to a final volume of 200 d in an UV-plate. The absorbance at 280 nm was measured prior to CRP analysis for calculation of protein recovery. DBS samples were analysed as single samples and blood references in duplicates in the CRP ELISA assay.

In the A280 measurement DBS samples and blood references in screening study part II were analysed as single samples. Blood references were analysed in duplicates in part I of the study.

CRP recovery Conc CR1' in sample Recovery % = ( )x 100 Conc CR1' in blood reference (average) Protein recovery The absorbance at 280 nm was used as a measure of overall protein recovery of the extracted samples.

Sample A280 Recovery% = ( )x100 Blood reference A280 (average) The first part of the prototype screening (table 3) contained prototypes soaked with F'icoll PM400, PM7O and PM2O. All soaked prototypes gave approximately 20% higher recovery compared to the reference paper.

Table 3 Average CRP recovery (triplleates) in DBS extract from Ficoll modified papers.

Paper Concentration in soaking solution CRP recovery (%) __________________________________________ (wt_96) __________________________________________ Plain 31 ETF paper (ic icrence) -81.2 3IETF+FicollPM400 7 _____________________ 102.6 31 ETF + Ficoll PM7O 7 ______________________ 98.5 3 1EIF + Ficoll PM70 5 100.5 3IETF + Ficoll PM2O 10 104.0 31ETF + Ficoll PM2O 7 ________________________ 105.1 3IETF + Ficoll PM2O 5 100.2 Table 4 Average CRP recovery (triplicates) in DBS extract from DBS paper prototypes.

Paper Conccnlralion in soaking/coupling CRP recovery (96) ___________________________________ solution (vt 96) ___________________________________ Plain 3IETF paper (reference) -73.4 Plain 903 paper (reference) -70.6 Handsheet from rcpulpcd 3 IETF, -61.1 treated with UGH ___________________________________ ___________________________________ 31ETF soakedw Ficoll PM7O 7 86.8 903 soaked w Ficoll PM7O 5 84.2 903 soakedw Ficoll PM7O 7 ______________________ 100.1 Handslicct florn repulped 31 ETF, 30 81.9 with UGH-coupled Ficoll PM7O ______________________________ ______________________________ Handsheet from repulped 3IETF, 50 ________________________ 84.8 with ECH-coupled Ficoll PM70 ____________________________ ____________________________ 3IETF soaked w CM Ficoll PM7O 5 92.1 3IETF soaked w CM Ficol! PM7O 7 _____________________ 93.0 3IETF with ECU-coupled CM 50 70.3 Ficoll PM7O ___________________________________ ___________________________________ 3lETFsoakedw DEAEFicoII 5 107.2 PM7O ____________________________ ____________________________ 3IETF soaked w DEAE Ficoll 7 102.4 PM70 ____________________________ ____________________________ 3IETF with ECH-eoupled DEAE 50 75.6 Ficoll PM7O ___________________________________ ___________________________________ 31 FITF soaked w DexUan 70 5 91.0 31ETF soakedw Dextran 70 7 90.1 3IETF with ECH-coupled Dextran 30 62.2

________________________________ _________________________________

3IETF with ECT-I-coup!ed Dextran 50 62.6

________________________________ _________________________________

3 1ETF soaked w Dextian 7 94.6 70/Ficoll PM7O 114 ________________________ ______________________________ 31 FIT soaked w Dextran 7 95.5 70/Ficoll PM7O 4:1 ________________________ ______________________________ The second part of the prototype screening (table 4) contained a wide variety of mainly Ficoll prototypes. Soaked prototypes gave also in this study clearly higher recovery compared with coupled prototypes in general. Also when coupling was done on the paper fibres an increased CRP recovery was obtained.

Mixtures ofFicoll and dextran also seem to have a stabilizing effect on the CRP molecule.

The ratio between Ficoll and dextran was not critical.

Even Ficoll prototypes with charged groups were Drking well in this study.

Prototypes soaked with charged Ficoll derivatives worked well in the ELISA application. For MS applications, the charged polymer may need to be removed from the sample first.

The recovery of total amount of protein (uot shown) was good from all prototypes and the variation seen reflects most likely the variation in spotted volume.

Example 4 -Stability of C-reactive protein, leptin and [52 Glycoprotein-1 in dried blood spots A forced stability study has been conducted of modified cellulose papers aimed for Dry Blood Spot (DBS) samples. DBS samples were spottcd on soaked and covalently coupled Ficoll prototypes to monitor stabilizing effect on C-reactivc protein (CRP), Leptin and 2 Glycoprotein-I after storage one week at -20 °C and 37 °C respectively. Commercial available ELISA kits have been used for evaluation. The goal was to find modification that is beneficial for more proteins than CRP, for which high recovery (compared to reference paper) has been established previously.

The result showed 10-20% higher recovery of CRP from soaked paper prototypes compared to prototypes modified by covalent coupling but no effect on leptin recovery. Soaked Ficoll PM400 was almost as good as untreated paper for f32GP1 and beneficial for both CRP and f32GP1 during storage at elevated temperature with less decrease in recovery compared to other prototypes.

To summarize the result it can be concluded that paper soaked with Ficoll PM400 seems beneficial for certain proteins upon storage as DBS.

Spotting and storage of DBS samples JIl blood aliquots were spotted on the different prototypes in triplicates. DBS were dried in RT for 4 h and the papers were then stored at 37°C in sealed bags with desiccant packets for one week. 15 gI blood aliquots were stored in 0.5 ml tubes for reference purpose (-20°C).

DBS extraction Spots were punched out (9 mm 0) and extracted in 24 well plates with 400 it1 buffer during I h in RT (mixing -500 rpm), +4°C overnight and I h RT (mixing -500 rpm). The plate was sealed during incubation and centrifuged at 3700 rpm in plate centrifuge prior to sample analyse. Sample for CRP and A280 analysis was removed and the sample plates were stored - 20°C.The extracted discs were removed before sample outtake for leptin and f32GP1 analysis.

CRP ELISA I A280 mcasurcmcnt The Human CRP ELISA Kit was used according to manufactures instruction except dilution recommendations (serum to be diluted 1:4000). Sample was diluted 1:270 as in previous studies.

Reference blood aliquots (n3) were diluted with 385 itl PBS-T. References and extracted samples were then further diluted 1:10 in CRP wash buffer to a final volume of 200 p.1 in an m'-p late.

The absorbance at 280 nm was measured prior to CRP analysis for calculation of protein recovery.

DBS samples were analysed as single samples and blood references in duplicates in the CRP EL1SA assay and A280 measurement Leptin ELISA The Human Leptin EL1SA Kit was used according to manufacturer's instruction exccpt dilution recommendations (serum to be diluted 1:3 in buffer supplied with kit). Since spiked sample another dilution need to be used and three dilutions were tested in a pre-study (1:50, 1:800 and 1:4000). 1:800 was chosen since the reference and sample ended up in the linear part of the standard curve. DBS samples were analysed as single samples and blood references in duplicates.

f32GPI ELISA The Human f32GP1 ELISA Kit was used according to manufacturer's instruction except dilution recommendations (serum to be diluted 1:5000). Three dilutions wcrc tested in a pre-study (1:50, 1:500 and 1:2500). Dilution 1:500 ended in the lower third of the standard curve and hence 1:250 was assumed to work well for the final study and hence used. DBS samples were analysed as single samples and blood references in duplicates.

CRP / leptin / 132GM recovery Conc CR/ leptin! 2GP1 in sample Recovery% ( )xlOO Conc CRP/ leptin / f32GP 1 in blood reference (average) Protein recovery The absorbancc at 250 nm was used as a measure of overall protein recovery of the extracted samples.

Sample A280 Recovery % = ( )x100 Blood reference A280 (average) The stability of three different proteins in DBS from paper prototypes have been monitored by ELISA. Table 2 shows a compressed summary of the result from the study.

Table 5. Average recovery (n=3) of three proteins obtained by extraction from DBS collected on DBS paper prototypes stored for one week at 37 C and analysed by ELISA. Recovery is calculated as % of amount obtained in blood reference sample (n=3, analysed in duplicate).

CRP Leptin Ji2 GP1 Protein DBS paper prototype recovery SWev recovery StDev recovery StDev recovery StDev 31 ETF 52,0 0,9 65,9 6,5 75,9 0,8 74,6 2,4 31 Efl fibre 51,1 1,7 57,9 1,5 76,8 1,3 77,2 2.5 PM2O 5% 55,1 6,7 60,5 9,9 85,7 12,4 77,2 6,0 P1"120 ECU 50% 55,7 5,3 53,8 5,7 88,6 11,6 87,1 14,9 PM7O 5% 67,1 3,4 68,2 4,2 70,0 15,8 76,9 2,6 PM7O ECU 50% 61,5 2,8 50,6 22,3 74,3 19,4 89,2 1,6 PM7O ECU fibre 64,3 4,7 64,8 2,2 83,5 15,0 88,0 6,9 PM400 5% 87,9 6,5 69,9 10,6 96,8 6,0 90,0 6,6 DEAE PM7O 5% 70,9 0,9 76,5 9,4 75,8 9,1 82,3 0,8 DEAE PM7O ECH 53,5 4,1 69,8 1,9 60,2 10,8 84,1 5,0 CM PM7O 5% 68,5 5,9 74,2 14,3 48,9 10,9 81,3 15,2 CM PM7O ECII 50% 60,9 0,8 66,9 9,4 69.6 1,9 88,2 5,4 Blood ref 100,0 5,7 100,0 10,4 100,0 8,9 100,0 9,4 Soaked prototypes gave as in the previous studies higher recovery of CRP. Ficoll ofhigher molecular size, PM7O and PM400 gave 25% higher CRP recovery for paper soaked with PM400 after storage one week at 37°C. Covalently modified paper shows best effect if the Ficoll was attached directly to the paper fibres. This may be due to higher degree of modification.

Some positive effect on recovery of leptin was found by the papers soaked with higher Mw Ficoll, although the uncertainty was higher in this ELISA, with standard deviations ranging from 2 to 22% within the triplicate results.

The standard deviation was large also in the 13201 assay (up to 27%). Ficoll PM400 gave higher recovery than the reference paper after storage at 37 °C indicating a stabilizing effect of this media.

ELISA assays for three proteins have been used for evaluation of stability during the DBS workflow. A high recovery has been assumed to reflect good stability during the interpretation of the result. However, depending on the antibody used in the assay even partially degraded and denatured proteins may contribute to the overall result and this has not been possible to control for. All ELISA kits used in thc study were aimed for serum samples.

Detailed evaluation of DBS recovery was difficult to obtain due to generally high standard deviation in the ELISA assays. Many steps are involved in the DBS workflow compared to doing the assay from serum samples.

To summarize the result it can be concluded that paper soaked with Ficoll PM400 is beneficial for certain proteins upon storage as DBS.

Soaked Ficoll PM400 was almost as good as untreated paper for f32GP1 and beneficial for both CRP and fi2GP 1 during storage at elevated temperature with less decrease in recovery compared to other prototypes.

Example S -Stability of alkaline phosphatase in dried blood spots Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in an alkaline environment. In this study the ALP activity was measured with substrate p-Nitrophenyl phosphate (pNPP). One product of the reaction, p-nitrophenol, exhibits yellow colour under alkaline conditions (maximal absorbance at 405nm). The rate of the reaction is directly proportional to the enzyme activity.

ALP activity was measured for prototypes with DBS stored at 37°C for a week Blood sample spiked with 20g/ml ALP: 15p1 of spiked blood are spotted on papers. Let the solutions air dry for at least 2 hours. Onc set of all 12 prototypc paper with DBS was stored at -20°C and the other set at 37°C for one week. 2 spots were made for each prototype and temperature.

After storage, the papers were let settle at room temperature. The spots were punched out as 10mm discs using a steel cork borer, hammer and a plastic cutting board. The disc was placed in a lOmL well of a LJniplate, and 400RL of the elution PBS-T buffer (PBS with 0.05% Tween 20) added. The plate was put on a shaker at 500 rpm for 1 hour, stored in refrigerator overnight and on a shaker again for 1 hour. For ALP activity measurements, 5R1 of the extracted solution were mixed with 195g1 of substrate pNPP solution and its absorbance at 4OSnm was followed.

Table 6. AkP activity recovery of extracted solutions from the prototypes.

Paper Concentration in soakingcoupling ALP activity recovery (94) ______________________________________ solution (wt 94) ______________________________________ Plain 31ETF paper (reference) -88 Handsheel from repuiped 3 1ETF -85 3 IETF soaked w DEAE FicolI 5 83 PM7O ______________________________ ______________________________ 3 IETF soaked w CM Ficoll PM7O 5 104 3IETF soaked w Ficoll PM2O 5 ________________________ 85 31 ETF soaked w Ficoll PM70 5 89 31ETF soakedwFico!I PM400 5 9S 3 IETF with ECH-coupled Ficoll 50 107 PM2O ________________________________ _________________________________ 31ETF with ECH-coupled Ficofl 50 99 PM7O ______________________ ____________________________ 31ETF with ECU-coupled DEAE 50 96 Ficoll PM7O ____________________________ ___________________________________ 31 ETF with ECU-coupled CM 50 92 Ficoll PM7O ___________________________________ ___________________________________ Handsheel from repulped 31 ETF, 50 95 with ECH-coup!cd Fico!! PM7O ___________________________________ ___________________________________ Best results on the ALP analysis were seen when using prototypes where Ficoll molecules were covalently coupled, the activity after storage at +37°C looks good for these prototypes, but also for the paper soaked with Ficoll PM400. These results indicate that the enzyme activity is good even after storage of the blood spotted papers at +37°C for one week.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any dcviccs or systems and pcrforming any incorporated mcthods. The patentable scopc of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims (1)

1. <claim-text>CLAIMS1. Paper fbr preservation of biological samples, having a surface weight of 40 -800 g/m2, comprising cellulose fibres or glass fibres, and further comprising 4-30 wt % of a hydmphilic branched carbohydrate polymer.</claim-text> <claim-text>2. The paper according to claim 1, comprising 10-25 wt% of the hydmphilic branched carbohydrate polymer.</claim-text> <claim-text>3. The paper according to claim 1 or 2, wherein the water soluble branched carbohydrate polymer has an avenge molecular weight of 15 -800 kDa.</claim-text> <claim-text>4. The paper according to any preceding claim, wherein the branched carbohydrate polymer has an average molecular weight of 70 -400 kDa, such as about 70 kDa or about 400 kDa.</claim-text> <claim-text>5. The paper according to any preceding claim, wherein said branched carbohydrate polymer comprises a copolymer of a mono-or disaccharide with a bifhnctional epoxide reagent 6. The paper according to any preceding claim, wherein said branched carbohydrate polymer is a sucrose-epichlorohydrin polymer, such as Ficoll.7. The paper according to any preceding claim, wherein a 10 wt % water solution of the branched carbohydrate polymer has a viscosity of I -10 mPas at 20°C.8. The paper according to any preceding claim, wherein the content of water extractables in said paper is 0-25 wt%, such as 0.1 -5 wt % or 3-20 wt %.9. The paper according to any preceding claim, further comprising 5-300 micmmole/g negatively or positively charged groups, such as carboxylate groups or amine groups.10. The paper according to any preceding claim, further comprising at least one dried biological sample, such as a dried blood sample.II. A sample collection device, comprising the paper according to any preceding claim.12. The sample collection device according to claim 11, wherein at least one sample application area is printed on the paper.13. A method for preservation of at least one biological sample, comprising the steps of: a) providing a biological sample, b) applying said biological sample on the paper of any preceding claim, and c) drying said paper with said biological sample.14. The method of claim 13, further comprising a step d) of storing the dried paper with the biological sample for at least one week.15. The method of claim 14, wherein in step d), the storage temperature is at least 0°C, such as 0-40°C or 20-40°C.16. The method of claims 13-15, further comprising a step e) of extracting at least one protein from said paper after storage and analyzing said protein.17. The method of claim 16, wherein said protein is a storage-sensitive protein 18. The method of claims 16 -17, wherein in step e) the recovery of said protein in a biologically active state is at least 60%, such as at least 80%.19. The method of claims 16-18, comprising in step e) analyzing said protein by an immunoassay or by mass spectrometry.20. A method of manufacturing a paper for preservation of biological samples, comprising the steps of a) providing cellulose fibres or glass fibres either in aqueous suspension or in the form of a base paper, b) contacting said fibres with a solution comprising 2-60 wt % of a water soluble branched carbohydrate polymer, c) if the fibres are in aqueous suspension, forming a paper sheet %m said fibres, and d) drying the paper.21. A method of manufacturing a paper for preservation of bio logical samples, comprising the steps of: a) providing a base paper having a surface weight of 40-800 g!m2 and comprising cellulose fibres or glass fibres, b) impregnating said base paper with a solution comprising 2-60 wt % of a water soluble branched carbohydrate polymer, and c) drying said impregnated paper.22. The method according to claim 20 or 21, wherein said branched carbohydrate polymer has an average molecular weight of 15 -800 kDa or 60 -500 kDa, such as about 70 kDa or about 400 kDa.23. The method according to any one of claims 20-22, wherein said branched carbohydrate polymer comprises a copolymer of a mono-or disaccharide with a bifunctional epoxide reagent 24. The method according to any one of claims 20-23, wherein said brauiched carbohydrate polymer is a sucrose-epichlorohydrin polymer, such as Ficoll.25. The method according to any one of claims 20-24, wherein said solution comprises 2 -60 wt %, such as 2 -15 wt% of said branched carbohydrate polymer 26. The method according to any one of claims 20-25, wherein the solution of thc branched carbohydrate polymer has a viscosity of I -20 mPas, such as I -10 mPas at 20°C.27. The method according to any one of claims 20-26, wherein the base paper comprise 5-300 micromole/g negatively charged groups, such as carboxylate groups, and wherein said negatively charged groups are not water extractable.28. The method according to any one of claims 20-27, wherein the fibres or the base paper are chemically activated and the method comprises a step of reacting the fibres or the base paper with the branched carbohydrate polymer.29. The method according to claim 28, wherein the activated fibres or base paper comprises epoxy groups.30. The method according to claim 28 or 29, further comprising a step of washing the fibres or the base paper after the reaction.</claim-text>